Abstract
To isolate cDNA clones encoding novel UGT2B enzymes, human prostate and LNCaP cell cDNA libraries were screened using a pool of steroid-specific UGT2B cDNA probes. In approximately 10(6) recombinants, we isolated 3 cDNA clones of 2.1 kilobases that encode a novel UGT2B enzyme. UGT2B17 is 95% identical with UGT2B15 and 91% identical with UGT2B8. Primary structure analysis of UGT2B17 based on the nucleotide sequence revealed a putative amino-terminal membrane insertion signal peptide, a carboxyl-terminal membrane-spanning region, and three potential asparagine-linked glycosylation sites. UGT2B17 cloned in the pBK-CMV expression vector was transfected into HK293 cells to obtain a stable clonal cell line expressing a high level of the active 53-kDa UGT2B17 enzyme. Of the over 60 endogenous and exogenous substances tested, 25 compounds revealed reactivity. The major substrates are eugenol > 4-methylumbelliferone > dihydrotestosterone > androstane-3alpha, 17beta-diol (3alpha-diol) > testosterone > androsterone (ADT). The apparent Km values obtained with tritiated steroids in intact cells were 0.4 microM for ADT, 0.7 microM for dihydrotestosterone, 1.0 microM for 3alpha-diol, and 3.4 microM for testosterone. Southern blot analysis of reverse transcription-polymerase chain reaction products revealed expression of UGT2B17 mRNA in various tissues including the liver, kidney, testis, uterus, placenta, mammary gland, adrenal gland, skin, and prostate. UGT2B17 is the first human uridine diphosphoglucuronosyltransferase enzyme expressed in extrahepatic tissues to have a specificity for ADT as well as testosterone, dihydrotestosterone, and 3alpha-diol.
Highlights
Uridine diphosphoglucuronosyltransferases (UGTs1; EC 2.4.1.17) represent a family of enzymes that catalyze the transfer of glucuronic acid from uridine diphosphoglucuronic acid to a wide variety of lipid-soluble drugs, environmental chemicals, and endogenous substances such as bilirubin, steroid hor
Isolation of a Novel UGT2B cDNA—Based on our previous observation that a significant amount of ADT-G is found in the human prostate [38], it was apparent that this tissue expresses a UGT capable of glucuronidating C19 steroids in the 3␣-hydroxyl position
Due to the high homology between the UGT2B cDNA clones isolated to date, which encode steroidspecific UGT enzymes, the libraries were screened using a pool of radiolabeled cDNA probes synthesized from the human UGT2B7, UGT2B10, and UGT2B15 cDNAs
Summary
Materials—UDP-glucuronic acid and all aglycons were obtained from Sigma and ICN Pharmaceutical Inc. (Montreal, Canada). CDNA Isolation—Affinity-purified benign prostate hyperplasia tissue and LNCaP cell mRNAs were used to construct cDNA libraries in the ZAP Express vector as specified by the supplier (Stratagene, La Jolla, CA). Both libraries were not amplified for screening where the filters were prehybridized in 40% formamide, 5 ϫ Denhardt’s solution, 5 ϫ SSPE, 0.1% SDS, and 100 mg/ml salmon sperm DNA for 4 h at 42 °C. Hybridization was carried out with the radiolabeled full-length UGT2B17 cDNA probe, and the blot was washed at high stringency conditions in 0.1 ϫ SSC, 0.1% SDS at 60 °C for 30 min and exposed for 4 h on an XAR hyperfilm. Plasma ADT-G and 3␣-diol-G were determined as described previously [24]
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